Anti-CD70 chimeric antigen receptors

ABSTRACT

The invention provides a chimeric antigen receptor (CAR) having antigenic specificity for CD70, the CAR comprising: an antigen binding-transmembrane domain comprising a CD27 amino acid sequence lacking all or a portion of the CD27 intracellular T cell signaling domain; a 4-1BB intracellular T cell signaling domain; a CD3ζ intracellular T cell signaling domain; and optionally, a CD28 intracellular T cell signaling domain. Nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the CARs are disclosed. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION APPLICATIONS

This patent application is a U.S. national stage of PCT/US2015/025047,filed Apr. 9, 2015, which claims the benefit of U.S. Provisional PatentApplication No. 62/088,882, filed Dec. 8, 2014, each of which isincorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under project numberZ01BC011337-04 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 55,212 Byte ASCII (Text) file named“728605_ST25.TXT,” dated May 22, 2017.

Cancer is a public health concern. Despite advances in treatments suchas chemotherapy, the prognosis for many cancers, including renal cellcarcinoma (RCC), glioblastoma, non-Hodgkin's lymphoma (NHL), chroniclymphocytic leukemia (CLL), diffuse large-B-cell lymphoma, andfollicular lymphoma, may be poor. Accordingly, there exists an unmetneed for additional treatments for cancer, particularly RCC,glioblastoma, NHL, CLL, diffuse large-B-cell lymphoma, and follicularlymphoma.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a chimeric antigen receptor(CAR) having antigenic specificity for CD70, the CAR comprising: anantigen binding-transmembrane domain comprising a CD27 amino acidsequence lacking all or a portion of the CD27 intracellular T cellsignaling domain, wherein the portion is at least amino acid residues237 to 260 as defined by SEQ ID NO: 2; a 4-1BB intracellular T cellsignaling domain; a CD3ζ (intracellular T cell signaling domain; andoptionally, a CD28 intracellular T cell signaling domain.

Another embodiment of the invention provides a CAR having antigenicspecificity for CD70 comprising an amino acid sequence at least about90% identical to any one of SEQ ID NOs: 11-13.

Further embodiments of the invention provide related nucleic acids,recombinant expression vectors, host cells, populations of cells, andpharmaceutical compositions relating to the CARs of the invention.

Additional embodiments of the invention provide methods of detecting thepresence of cancer in a mammal and methods of treating or preventingcancer in a mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A and 1B are graphs showing the tumor size (mm²) of B16/mCD70-(A)or B16-(B) tumor bearing mice over a period of time (days) followingadministration of mCD27-CD3ζ CAR-transduced cells (closed circles),untransduced cells (open circles), phosphate buffered saline (PBS) (×),or pmel+VI (squares) and irradiation (500 Rads).

FIGS. 1C and 1D are graphs showing the tumor size (mm²) ofB16/mCD70-tumor bearing mice over a period of time (days) followingadministration of mCD27-CD3ζ CAR-transduced cells at a dose of 1−10⁴(▾), 1×10⁵ (closed squares), 1×10⁶ (▴), or 1×10⁷ (closed circles) cellsper mouse; PBS (open squares); cells transduced with an empty vector(open circles); or pmel+VI (diamonds) with (C) or without (D)irradiation (500 Rads).

FIG. 1E is a graph showing the survival (%) of B16/mCD70-tumor bearingmice over a period of time (days) following administration of mCD27-CD3ζCAR-transduced cells at a dose of 1×10⁴ (diamonds), 1×10⁵ (▾), 1×10⁶(▴), or 1×10⁷ (closed circles) cells per mouse; PBS (open squares);cells transduced with an empty vector (open circles); or pmel+VI (Δ),followed by irradiation (500 Rads).

FIG. 1F is a graph showing the tumor size (mm²) of B16/mCD70-tumorbearing mice over a period of time (days) following administration ofmCD27-CD3ζ CAR-transduced cells (squares), untransduced cells (Δ), cellstransduced with an empty vector (∇), or pmel+VI (circles), followed byirradiation and administration of IL-2.

FIGS. 2A-2D are graphs showing the average weight (g) of B16/mCD70-(Aand B) or B16-(C and D) tumor bearing mice over a period of time (days)following administration of mCD27-CD3ζ CAR-transduced cells (closedcircles), untransduced cells (open squares), phosphate buffered saline(PBS) (∇), or pmel+VI (Δ) with (A and C) or without (B and D)irradiation (500 Rads).

FIGS. 2E-2H are graphs showing the absolute white blood cell count(K/μl) (E and F) or splenocyte count (×10⁷ per spleen) (G and H) ofB16/mCD70-tumor bearing mice over a period of time (days) followingadministration of mCD27-CD3ζ CAR-transduced cells (cross-hatched bars),untransduced cells (unshaded bars), or cells transduced with a vectorencoding green fluorescent protein (GFP) (diagonally striped bars) with(E and G) or without (F and H) irradiation (500 Rads).

FIG. 2I is a graph showing serum interferon (IFN) gamma (pg/ml) levelsof B16/mCD70-tumor bearing mice over a period of time (days) followingadministration of mCD27-CD3ζ CAR-transduced cells with (black bars) orwithout (horizontally striped bars) irradiation or cells transduced witha vector encoding GFP with (checkered bars) or without (unshaded bars)irradiation (500 Rads).

FIG. 3 is a graph showing IFN-γ (pg/ml) secreted upon culture of human Tcells transduced with an empty retroviral vector (control) (MSGV1) orone of fCD27-CD3ζ (SEQ ID NO: 7), ΔCD27-CD28-CD3ζ ((SEQ ID NO: 8),ΔCD27-4-1BB-CD3ζ ((SEQ ID NO: 9), ΔCD27-CD28-4-1BB-CD3ζ ((SEQ ID NO:10), fCD27-CD28-CD3ζ (SEQ ID NO: 11), fCD27-4-1BB-CD3ζ (SEQ ID NO: 12),or fCD27-CD28-4-1BB-CD3ζ (SEQ ID NO: 13) alone (medium) (verticallystriped bars) or upon co-culture with control target cells 624 mel(checkered bars), 624/CD70 (black bars), 938 mel (dotted bars), or938/CD70 (white bars) or RCC target cells RCC 2245R (forward slashedbars), RCC 2246R (backslashed bars), RCC 2361R (boxed bars), or RCC 1764(herringbone bars).

FIG. 4 is a graph showing IFN-γ (pg/ml) secreted upon culture ofuntransduced (UT) cells or retroviral packaging clone A2, A10, B3, C1,E3, or G2 transduced with ΔCD27-4-1BB-CD3ζ ((SEQ ID NO: 9) alone(medium, vertically striped bars) or upon co-culture with control targetcells SNU1079 (dotted bars), SNU1196 (white bars), 938 mel (checkeredbars), or 938/CD70 (black bars) or RCC target cells RCC 2245R (forwardslashed bars), RCC 2246R (backslashed bars), RCC 2361R (boxed bars), orRCC 1764 (herringbone bars).

FIG. 5 is a graph showing IFN-γ (pg/ml) secreted upon culture ofuntransduced (UT) cells or retroviral packaging clone A2, B11, C5, or D2transduced with ΔCD27-4-1BB-CD3ζ (SEQ ID NO: 9) alone (medium,vertically striped bars) or upon co-culture with control target cellsSNU1079 (dotted bars), SNU1196 (white bars), 938 mel (checkered bars),or 938/CD70 (black bars) or RCC target cells RCC 2245R (forward slashedbars), RCC 2246R (backslashed bars), RCC 2361R (boxed bars), or RCC 1764(herringbone bars).

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a chimeric antigen receptor(CAR) having antigenic specificity for CD70, the CAR comprising: anantigen binding-transmembrane domain comprising a CD27 amino acidsequence lacking all or a portion of the CD27 intracellular T cellsignaling domain, wherein the portion is at least amino acid residues237 to 260 as defined by SEQ ID NO: 2; a 4-1BB intracellular T cellsignaling domain; a CD3ζ (intracellular T cell signaling domain; andoptionally, a CD28 intracellular T cell signaling domain. Hereinafter,references to a “CAR” also refer to functional portions and functionalvariants of the CAR, unless specified otherwise.

A CAR is an artificially constructed hybrid protein or polypeptidecontaining the antigen binding domains of a receptor (e.g., a tumornecrosis factor (TNF) receptor) linked to T-cell signaling domains.Characteristics of CARs include their ability to redirect T-cellspecificity and reactivity toward a selected target in a non-majorhistocompatibility complex (MHC)-restricted manner, exploiting theantigen-binding properties of receptors. The non-MHC-restricted antigenrecognition gives T cells expressing CARs the ability to recognizeantigen independent of antigen processing, thus bypassing a majormechanism of tumor escape. Moreover, when expressed in T-cells, CARsadvantageously do not dimerize with endogenous T cell receptor (TCR)alpha and beta chains.

The phrases “have antigen(ic) specificity” and “elicit antigen-specificresponse,” as used herein, means that the CAR can specifically bind toand immunologically recognize an antigen, such that binding of the CARto the antigen elicits an immune response.

The CARs of the invention have antigen specificity for CD70. CD70belongs to the TNF superfamily and has the amino acid sequence of SEQ IDNO: 1 CD70 is a costimulatory molecule that is involved in theproliferation and survival of lymphoid-derived cells when it interactswith its receptor, CD27. Normal, non-cancerous expression of CD70 isrestricted to lymphoid tissues such as activated T cells, B cells,natural killer (NK) cells, monocytes, and dendritic cells. CD70 isexpressed in a variety of human cancers such as, for example, RCC(Diegmann et al., Eur. J. Cancer, 41: 1794-801 (2005)) (for example,clear cell RCC (ccRCC)), glioblastoma (Held-Feindt et al., Int. J.Cancer, 98: 352-56 (2002); Wischhusen et al., Cancer Res., 62: 2592-99(2002)), NHL and CLL (Lens et al., Br. J. Haematol., 106: 491-503(1999)), diffuse large-B-cell lymphoma, and follicular lymphoma.

Without being bound to a particular theory or mechanism, it is believedthat by eliciting an antigen-specific response against CD70, theinventive CARs provide for one or more of any of the following:targeting and destroying CD70-expressing cancer cells, reducing oreliminating cancer cells, facilitating infiltration of immune cells totumor site(s), and enhancing/extending anti-cancer responses. Becausenormal CD70 expression is limited to lymphoid tissues such as activatedT cells, B cells, NK cells, monocytes, and dendritic cells, it iscontemplated that the inventive CARs advantageously substantially avoidtargeting/destroying many normal tissues.

An embodiment of the invention provides a CAR comprising an antigenbinding-transmembrane domain comprising a CD27 amino acid sequence. Inthis regard, the CAR may comprise both a CD27 antigen binding domain anda CD27 transmembrane domain. The CD27 may comprise or consist of anysuitable human antigen binding-transmembrane domain CD27 amino acidsequence. In an embodiment of the invention, full-length CD27, includingthe antigen binding domain, the transmembrane domain, and theintracellular T cell signaling domain, has the amino acid sequence ofSEQ ID NO: 2. In an embodiment of the invention, the antigen bindingdomain of CD27 is composed of amino acid residues 1-188 of SEQ ID NO: 2and has the amino acid sequence of SEQ ID NO: 21, the transmembranedomain of CD27 is composed of amino acid residues 189-211 of SEQ ID NO:2 and has the amino acid sequence of SEQ ID NO: 22, and theintracellular T cell signaling domain of CD27 is composed of amino acidresidues 212-260 of SEQ ID NO: 2 and has the amino acid sequence of SEQID NO: 23. Accordingly, in an embodiment of the invention, the CARcomprises an antigen binding-transmembrane domain comprising the aminoacid sequences of SEQ ID NOs: 21 and 22. The antigen binding domain ofCD27 specifically binds to CD70.

An embodiment of the invention provides a CAR comprising an antigenbinding-transmembrane domain comprising a CD27 amino acid sequencelacking all or a portion of the CD27 intracellular T cell signalingdomain, wherein the portion that is lacking from the CAR is at leastcontiguous amino acid residues 237 to 260, at least contiguous aminoacid residues 236 to 260, at least contiguous amino acid residues 235 to260, at least contiguous amino acid residues 234 to 260, at leastcontiguous amino acid residues 233 to 260, at least contiguous aminoacid residues 232 to 260, at least contiguous amino acid residues 231 to260, at least contiguous amino acid residues 230 to 260, at leastcontiguous amino acid residues 229 to 260, at least contiguous aminoacid residues 228 to 260, at least contiguous amino acid residues 227 to260, at least contiguous amino acid residues 226 to 260, at leastcontiguous amino acid residues 225 to 260, at least contiguous aminoacid residues 224 to 260, at least contiguous amino acid residues 223 to260, at least contiguous amino acid residues 222 to 260, at leastcontiguous amino acid residues 221 to 260, at least contiguous aminoacid residues 220 to 260, at least contiguous amino acid residues 219 to260, at least contiguous amino acid residues 218 to 260, at leastcontiguous amino acid residues 217 to 260, at least contiguous aminoacid residues 216 to 260, at least contiguous amino acid residues 215 to260, at least contiguous amino acid residues 214 to 260, or at leastcontiguous amino acid residues 213 to 260, as defined by SEQ ID NO: 2.In an embodiment of the invention, the antigen binding-transmembranedomain comprises a CD27 amino acid sequence lacking contiguous aminoacid residues 237 to 260, contiguous amino acid residues 236 to 260,contiguous amino acid residues 235 to 260, contiguous amino acidresidues 234 to 260, contiguous amino acid residues 233 to 260,contiguous amino acid residues 232 to 260, contiguous amino acidresidues 231 to 260, contiguous amino acid residues 230 to 260,contiguous amino acid residues 229 to 260, contiguous amino acidresidues 228 to 260, contiguous amino acid residues 227 to 260,contiguous amino acid residues 226 to 260, contiguous amino acidresidues 225 to 260, contiguous amino acid residues 224 to 260,contiguous amino acid residues 223 to 260, contiguous amino acidresidues 222 to 260, contiguous amino acid residues 221 to 260,contiguous amino acid residues 220 to 260, contiguous amino acidresidues 219 to 260, contiguous amino acid residues 218 to 260,contiguous amino acid residues 217 to 260, contiguous amino acidresidues 216 to 260, contiguous amino acid residues 215 to 260,contiguous amino acid residues 214 to 260, or contiguous amino acidresidues 213 to 260, of SEQ ID NO: 2. A CD27 amino acid sequence lackingall or a portion of the CD27 intracellular T cell signaling domain isalso referred to herein as a “truncated CD27 amino acid sequence” or a“truncated CD27.”

In a preferred embodiment, the antigen binding-transmembrane domaincomprises a CD27 amino acid sequence lacking all of the CD27intracellular T cell signaling domain. In this regard, the antigenbinding-transmembrane domain comprises a CD27 amino acid sequencelacking contiguous amino acid residues 212 to 260 as defined by SEQ IDNO: 2 or a CD27 amino acid sequence lacking contiguous amino acidresidues 212 to 260 of SEQ ID NO: 2. In an embodiment of the invention,the antigen binding-transmembrane domain comprises a CD27 amino acidsequence lacking the amino acid sequence of SEQ ID NO: 23. In anembodiment of the invention, the antigen binding-transmembrane domainthat comprises a CD27 amino acid sequence lacking all of the CD27intracellular T cell signaling domain comprises or consists of an aminoacid sequence at least about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99% identicalto SEQ ID NO: 3 or which comprises or consists of the amino acidsequence of SEQ ID NO: 3.

The CAR may further comprise a 4-1BB intracellular T cell signalingdomain; a CD3 zeta (ζ) intracellular T cell signaling domain; andoptionally, a CD28 intracellular T cell signaling domain. In anembodiment of the invention, the CAR comprises comprising a 4-1BBintracellular T cell signaling domain, a CD3ζ intracellular T cellsignaling domain, and a CD28 intracellular T cell signaling domain. Inanother embodiment of the invention, the CAR comprises a 4-1BBintracellular T cell signaling domain and a CD3ζ (intracellular T cellsignaling domain. In a preferred embodiment, the 4-1BB, CD3ζ, and CD28intracellular T cell signaling domains are human. CD28 is a T cellmarker important in T cell co-stimulation. 4-1BB, also known as CD137,transmits a potent costimulatory signal to T cells, promotingdifferentiation and enhancing long-term survival of T lymphocytes. CD3ζassociates with TCRs to produce a signal and contains immunoreceptortyrosine-based activation motifs (ITAMs).

The CD3ζ intracellular T cell signaling domain may comprise or consistof any suitable human CD3ζ intracellular T cell signaling domain aminoacid sequence. In an embodiment of the invention, the CD3ζ intracellularT cell signaling domain comprises or consists of an amino acid sequenceat least about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ IDNO: 4. Preferably, the CD3ζ (intracellular T cell signaling domaincomprises or consists of the amino acid sequence of SEQ ID NO: 4.

The 4-1BB intracellular T cell signaling domain may comprise or consistof any suitable human 4-1BB intracellular T cell signaling domain aminoacid sequence. In an embodiment of the invention, the 4-1BBintracellular T cell signaling domain comprises or consists of an aminoacid sequence at least about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99% identicalto SEQ ID NO: 5. Preferably, the 4-1BB intracellular T cell signalingdomain comprises or consists of the amino acid sequence of SEQ ID NO: 5.

The CD28 intracellular T cell signaling domain may comprise or consistof any suitable human CD28 intracellular T cell signaling domain aminoacid sequence. In an embodiment of the invention, the CD28 intracellularT cell signaling domain comprises or consists of an amino acid sequenceat least about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ IDNO: 6. Preferably, the CD28 intracellular T cell signaling domaincomprises or consists of the amino acid sequence of SEQ ID NO: 6.

In an embodiment of the invention, the CAR comprises a full-length CD27amino acid sequence, including a CD27 antigen binding domain, a CD27transmembrane domain, and a CD27 intracellular T cell signaling domain,in combination with a CD3ζ (intracellular T cell signaling domain (fulllength (f)CD27-CD3ζ CAR). In this regard, the CAR may comprise orconsist of a full-length CD27 amino acid sequence at least about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to SEQ ID NO: 2 and any of theCD3ζ amino acid sequences described herein with respect to other aspectsof the invention. For example, the fCD27-CD3ζ CAR may comprise orconsist of the full-length CD27 amino acid sequence of SEQ ID NO: 2 andthe CD3ζ amino acid sequence of SEQ ID NO: 4. In an embodiment of theinvention, the fCD27-CD3ζ (CAR may comprise or consist of an amino acidsequence at least about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% identical toSEQ ID NO: 7. Preferably, the fCD27-CD3ζ (CAR comprises or consists ofthe amino acid sequence of SEQ ID NO: 7. In an embodiment of theinvention, the fCD27-CD3ζ (CAR lacks one or both of truncated CD19 andDsRed.

In an embodiment of the invention, the CAR comprises a full-length CD27amino acid sequence, including a CD27 antigen binding domain, a CD27transmembrane domain, and a CD27 intracellular T cell signaling domain,in combination with a CD3ζ intracellular T cell signaling domain and aCD28 intracellular T cell signaling domain (fCD27-CD28-CD3ζ). In thisregard, the CAR may comprise or consist of any of the full-length CD27amino acid sequences, any of the CD3ζ amino acid sequences, and any ofthe CD28 amino acid sequences described herein with respect to otheraspects of the invention. For example, the fCD27-CD28-CD3ζ CAR maycomprise or consist of the full-length CD27 amino acid sequence of SEQID NO: 2, the CD3ζ (amino acid sequence of SEQ ID NO: 4, and the CD28amino acid sequence of SEQ ID NO: 6. In an embodiment of the invention,the fCD27-CD28-CD3ζ CAR may comprise or consist of an amino acidsequence at least about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% identical toSEQ ID NO: 11. Preferably, the fCD27-CD28-CD3ζ (CAR comprises orconsists of the amino acid sequence of SEQ ID NO: 11.

In an embodiment of the invention, the CAR comprises a full-length CD27amino acid sequence, including a CD27 antigen binding domain, a CD27transmembrane domain, and a CD27 intracellular T cell signaling domain,in combination with a CD3ζ intracellular T cell signaling domain and a4-1BB intracellular T cell signaling domain (fCD27-4-1BB-CD3ζ). In thisregard, the CAR may comprise or consist of any of the full-length CD27amino acid sequences, any of the CD3ζ (amino acid sequences, and any ofthe 4-1BB amino acid sequences described herein with respect to otheraspects of the invention. For example, the fCD27-4-1BB-CD3ζ CAR maycomprise or consist of the full-length CD27 amino acid sequence of SEQID NO: 2, the CD3ζ (amino acid sequence of SEQ ID NO: 4, and the 4-1BBamino acid sequence of SEQ ID NO: 5. In an embodiment of the invention,the fCD27-4-1BB-CD3ζ(CAR may comprise or consist of an amino acidsequence at least about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% identical toSEQ ID NO: 12. Preferably, the fCD27-4-1BB-CD3ζ (CAR comprises orconsists of the amino acid sequence of SEQ ID NO: 12.

In an embodiment of the invention, the CAR comprises a full-length CD27amino acid sequence, including a CD27 antigen binding domain, a CD27transmembrane domain, and a CD27 intracellular T cell signaling domain,in combination with a CD3ζ (intracellular T cell signaling domain, a4-1BB intracellular T cell signaling domain, and a CD28 intracellularsignaling domain (fCD27-CD28-4-1BB-CD3ζ). In this regard, the CAR maycomprise or consist of any of the full-length CD27 amino acid sequences,any of the CD3ζ amino acid sequences, any of the 4-1BB amino acidsequences, and any of the CD28 amino acid sequences described hereinwith respect to other aspects of the invention. For example, thefCD27-CD28-4-1BB-CD3ζ CAR may comprise or consist of the full-lengthCD27 amino acid sequence of SEQ ID NO: 2, the CD3ζ (amino acid sequenceof SEQ ID NO: 4, the 4-1BB amino acid sequence of SEQ ID NO: 5, and theCD28 amino acid sequence of SEQ ID NO: 6. In an embodiment of theinvention, the fCD27-CD28-4-1BB-CD3ζ CAR may comprise or consist of anamino acid sequence at least about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 13. Preferably, the fCD27-CD28-4-1BB-CD3ζ CARcomprises or consists of the amino acid sequence of SEQ ID NO: 13.

In an embodiment of the invention, the CAR comprises a full-length mouseCD27 amino acid sequence, including a CD27 antigen binding domain, aCD27 transmembrane domain, and a CD27 intracellular T cell signalingdomain, in combination with a mouse CD3ζ intracellular T cell signalingdomain (mCD27-CD3ζ). In this regard, the CAR may comprise or consist ofa full-length mouse CD27 amino acid sequence at least about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99% identical to SEQ ID NO: 26 in combination with amouse CD3ζ amino acid sequence at least about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, orabout 99% identical to SEQ ID NO: 27. For example, the mCD27-CD3ζ (CARmay comprise or consist of the full-length mouse CD27 amino acidsequence of SEQ ID NO: 26 and the mouse CD3ζ (amino acid sequence of SEQID NO: 27. In an embodiment of the invention, the mCD27-CD3ζ (CAR maycomprise or consist of an amino acid sequence at least about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99% identical to SEQ ID NO: 25. Preferably, themCD27-CD3ζ CAR comprises or consists of the amino acid sequence of SEQID NO: 25.

In an embodiment of the invention, the CAR comprises an antigenbinding-transmembrane domain comprising a truncated CD27 amino acidsequence which lacks all of the CD27 intracellular T cell signalingdomain, in combination with a CD3ζ intracellular T cell signaling domainand a CD28 intracellular T cell signaling domain (truncated (Δ)CD27-CD28-CD3ζ). In this regard, the CAR may comprise or consist of atruncated CD27 antigen binding-transmembrane domain amino acid sequenceat least about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ IDNO: 3 in combination with any of the CD3ζ intracellular T cell signalingdomain amino acid sequences and any of the CD28 intracellular T cellsignaling domain amino acid sequences described herein with respect toother aspects of the invention. For example, the ΔCD27-CD28-CD3ζ (CARmay comprise or consist of the truncated CD27 antigenbinding-transmembrane domain amino acid sequence of SEQ ID NO: 3, theCD3ζ amino acid sequence of SEQ ID NO: 4, and the CD28 amino acidsequence of SEQ ID NO: 6. In an embodiment of the invention, theΔCD27-CD28-CD3ζ (CAR may comprise or consist of an amino acid sequenceat least about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ IDNO: 8. Preferably, the ΔCD27-CD28-CD3ζ CAR comprises or consists of theamino acid sequence of SEQ ID NO: 8.

In an embodiment of the invention, the CAR comprises an antigenbinding-transmembrane domain comprising a truncated CD27 amino acidsequence which lacks all of the CD27 intracellular T cell signalingdomain, in combination with a CD3ζ intracellular T cell signaling domainand a 4-1BB intracellular T cell signaling domain (ΔCD27-4-1BB-CD3ζ). Inthis regard, the CAR may comprise or consist of any of the truncatedCD27 antigen binding-transmembrane domain amino acid sequences, any ofthe CD3ζ intracellular T cell signaling domain amino acid sequences, andany of the 4-1BB intracellular T cell signaling domain amino acidsequences described herein with respect to other aspects of theinvention. For example, the ΔCD27-4-1BB-CD3ζ CAR may comprise or consistof the truncated CD27 antigen binding-transmembrane domain amino acidsequence of SEQ ID NO: 3, the CD3ζ (amino acid sequence of SEQ ID NO: 4,and the 4-1BB amino acid sequence of SEQ ID NO: 5. In an embodiment ofthe invention, the ΔCD27-4-1BB-CD3ζ (CAR may comprise or consist of anamino acid sequence at least about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 9. Preferably, the ΔCD27-4-1BB-CD3ζ CARcomprises or consists of the amino acid sequence of SEQ ID NO: 9.

In an embodiment of the invention, the CAR comprises an antigenbinding-transmembrane domain comprising a truncated CD27 amino acidsequence which lacks all of the CD27 intracellular T cell signalingdomain, in combination with a CD3ζ intracellular T cell signalingdomain, a CD28 intracellular T cell signaling domain, and a 4-1BBintracellular T cell signaling domain (ΔCD27-CD28-4-1BB-CD3ζ). In thisregard, the CAR may comprise or consist of any of the truncated CD27antigen binding—transmembrane domain amino acid sequences, any of theCD3ζ intracellular T cell signaling domain amino acid sequences, any ofthe CD28 intracellular T cell signaling domain amino acid sequences, andany of the 4-1BB intracellular T cell signaling domain amino acidsequences described herein with respect to other aspects of theinvention. For example, the ΔCD27-CD28-4-1BB-CD3ζ (CAR may comprise orconsist of the truncated CD27 antigen binding-transmembrane domain aminoacid sequence of SEQ ID NO: 3, the CD3ζ amino acid sequence of SEQ IDNO: 4, the CD28 amino acid sequence of SEQ ID NO: 6, and the 4-1BB aminoacid sequence of SEQ ID NO: 5. In an embodiment of the invention, theΔCD27-CD28-4-1BB-CD3ζ (CAR may comprise or consist of an amino acidsequence at least about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% identical toSEQ ID NO: 10. Preferably, the ΔCD27-CD28-4-1BB-CD3ζ (CAR comprises orconsists of the amino acid sequence of SEQ ID NO: 10.

In an embodiment of the invention, the CAR comprises an amino acidsequence at least about 90% identical to any one of SEQ ID NOs: 8-10. Inan embodiment of the invention, the CAR comprises an amino acid sequenceat least about 90% identical to SEQ ID NO: 9 or 10. In anotherembodiment of the invention, the CAR comprises an amino acid sequence atleast about 90% identical to any one of SEQ ID NOs: 11-13. In anotherembodiment of the invention, the CAR comprises an amino acid sequence atleast about 90% identical to SEQ ID NO: 12 or 13. Preferably, the CARcomprises, consists of, or consists essentially of any one of the aminoacid sequences set forth in Table 1A. In a preferred embodiment of theinvention, the CAR comprises the amino acid sequence of any one of SEQID NOs: 7-13. Preferably, the CAR comprises the amino acid sequence ofany one of SEQ ID NO: 9, 10, 12, and 13.

TABLE 1A Antigen binding and Transmembrane Intracellular T Cell CARDomain Signaling Domain full length (f) CD27-CD3ζ (SEQ full length humanCD27 and ID NO: 7) human CD27 human CD3ζ truncated (Δ) CD27-CD28-truncated human CD28 and CD3ζ (SEQ ID NO: 8) human CD27 human CD3ζΔCD27-4-1BB-CD3ζ (SEQ ID truncated human 4-1BB and NO: 9) human CD27human CD3ζ ΔCD27-CD28-4-1BB-CD3ζ truncated human 4-1BB (SEQ ID NO: 10)human CD27 human CD28 and human CD3ζ fCD27-CD28-CD3ζ (SEQ ID full-lengthhuman CD27 NO: 11) human CD27 human CD28 and human CD3ζ fCD27-4-1BB-CD3ζ(SEQ ID full-length human CD27 NO: 12) human CD27 human 4-1BB and humanCD3ζ fCD27-CD28-4-1BB-CD3ζ full-length human CD27 (SEQ ID NO: 13) humanCD27 human 4-1BB human CD28 and human CD3ζ mCD27-CD3ζ (SEQ ID NO: 25)full length mouse CD3ζ mouse CD27

Included in the scope of the invention are functional portions of theinventive CARs described herein. The term “functional portion” when usedin reference to a CAR refers to any part or fragment of the CAR of theinvention, which part or fragment retains the biological activity of theCAR of which it is a part (the parent CAR). Functional portionsencompass, for example, those parts of a CAR that retain the ability torecognize target cells, or detect, treat, or prevent cancer, to asimilar extent, the same extent, or to a higher extent, as the parentCAR. In reference to the parent CAR, the functional portion cancomprise, for instance, about 10%, about 25%, about 30%, about 50%,about 68%, about 80%, about 90%, about 95%, or more, of the parent CAR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent CAR.Desirably, the additional amino acids do not interfere with thebiological function of the functional portion, e.g., recognize targetcells, detect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity, as compared tothe biological activity of the parent CAR.

Included in the scope of the invention are functional variants of theinventive CARs described herein. The term “functional variant” as usedherein refers to a CAR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent CAR, whichfunctional variant retains the biological activity of the CAR of whichit is a variant. Functional variants encompass, for example, thosevariants of the CAR described herein (the parent CAR) that retain theability to recognize target cells to a similar extent, the same extent,or to a higher extent, as the parent CAR. In reference to the parentCAR, the functional variant can, for instance, be at least about 30%,about 50%, about 75%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% ormore identical in amino acid sequence to the parent CAR.

A functional variant can, for example, comprise the amino acid sequenceof the parent CAR with at least one conservative amino acidsubstitution. Alternatively or additionally, the functional variants cancomprise the amino acid sequence of the parent CAR with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parent CAR.

Amino acid substitutions of the inventive CARs are preferablyconservative amino acid substitutions. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the same orsimilar chemical or physical properties. For instance, the conservativeamino acid substitution can be an acidic/negatively charged polar aminoacid substituted for another acidic/negatively charged polar amino acid(e.g., Asp or Glu), an amino acid with a nonpolar side chain substitutedfor another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val,Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positivelycharged polar amino acid substituted for another basic/positivelycharged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged aminoacid with a polar side chain substituted for another uncharged aminoacid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), anamino acid with a beta-branched side-chain substituted for another aminoacid with a beta-branched side-chain (e.g., Ile, Thr, and Val), an aminoacid with an aromatic side-chain substituted for another amino acid withan aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

The CAR can consist essentially of the specified amino acid sequence orsequences described herein, such that other components, e.g., otheramino acids, do not materially change the biological activity of thefunctional variant.

The CARs of embodiments of the invention can be of any length, i.e., cancomprise any number of amino acids, provided that the CARs retain theirbiological activity, e.g., the ability to specifically bind to antigen,detect cancer cells in a mammal, or treat or prevent cancer in a mammal,etc. For example, the CAR can be about 50 to about 5000 amino acidslong, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600,700, 800, 900, 1000 or more amino acids in length.

The CARs of embodiments of the invention can comprise synthetic aminoacids in place of one or more naturally-occurring amino acids. Suchsynthetic amino acids are known in the art, and include, for example,aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid,homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The CARs of embodiments of the invention can be glycosylated, amidated,carboxylated, phosphorylated, esterified, N-acylated, cyclized via,e.g., a disulfide bridge, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated.

The CARs of embodiments of the invention can be obtained by methodsknown in the art such as, for example, de novo synthesis. Also,polypeptides and proteins can be recombinantly produced using thenucleic acids described herein using standard recombinant methods. See,for instance, Green and Sambrook, Molecular Cloning: A LaboratoryManual, 4^(th) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(2012). Alternatively, the CARs described herein can be commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the inventive CARs can besynthetic, recombinant, isolated, and/or purified.

Further provided by an embodiment of the invention is a nucleic acidcomprising a nucleotide sequence encoding any of the CARs describedherein. The nucleic acids of the invention may comprise a nucleotidesequence encoding any of the antigen binding domains, transmembranedomains, and/or intracellular T cell signaling domains described herein.In an embodiment of the invention, the nucleic acid comprises, consistsof, or consists essentially of any one of the nucleotide sequences setforth in Table 1B. Preferably, the nucleic acid comprises the nucleotidesequence of any one of SEQ ID NOs: 14-20. Preferably, the nucleic acidcomprises the nucleotide sequence of any one of SEQ ID NOs: 16, 17, 19,and 20. In an embodiment of the invention, the nucleotide sequenceencoding the fCD27-CD3ζ CAR does not encode one or both of truncatedCD19 and DsRed.

TABLE 1B Antigen binding and Transmembrane Intracellular T Cell CARDomain Signaling Domain full length (f) CD27-CD3ζ (SEQ full length humanCD27 and ID NO: 14) human CD27 human CD3ζ truncated (Δ) CD27-CD28-truncated human CD28 and CD3ζ (SEQ ID NO: 15) human CD27 human CD3ζΔCD27-4-1BB-CD3ζ (SEQ ID truncated human 4-1BB and NO: 16) human CD27human CD3ζ ΔCD27-CD28-4-1BB-CD3ζ truncated human 4-1BB (SEQ ID NO: 17)human CD27 human CD28 and human CD3ζ fCD27-CD28-CD3ζ (SEQ ID full-lengthhuman CD27 NO: 18) human CD27 human CD28 and human CD3ζ fCD27-4-1BB-CD3ζ(SEQ ID full-length human CD27 NO: 19) human CD27 human 4-1BB and humanCD3ζ fCD27-CD28-4-1BB-CD3ζ full-length human CD27 (SEQ ID NO: 20) humanCD27 human 4-1BB human CD28 and human CD3ζ mCD27-CD3ζ (SEQ ID NO: 24)full length mouse CD3ζ mouse CD27

“Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In some embodiments, the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions. In someembodiments, the nucleic acid may encode additional amino acid sequencesthat do not affect the function of the CAR and which may or may not betranslated upon expression of the nucleic acid by a host cell.

The nucleic acids of an embodiment of the invention may be recombinant.As used herein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

A recombinant nucleic acid may be one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques, such as thosedescribed in Green and Sambrook, supra. The nucleic acids can beconstructed based on chemical synthesis and/or enzymatic ligationreactions using procedures known in the art. See, for example, Green andSambrook, supra. For example, a nucleic acid can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedupon hybridization (e.g., phosphorothioate derivatives and acridinesubstituted nucleotides). Examples of modified nucleotides that can beused to generate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methyl guanine, 3-methyl cytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any isolated or purified nucleotidesequence which encodes any of the CARs described herein with respect toother aspects of the invention. Alternatively, the nucleotide sequencecan comprise a nucleotide sequence which is degenerate to any of thesequences or a combination of degenerate sequences.

An embodiment of the invention also provides an isolated or purifiednucleic acid comprising a nucleotide sequence which is complementary tothe nucleotide sequence of any of the nucleic acids described herein ora nucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditions mayhybridize under high stringency conditions. By “high stringencyconditions” is meant that the nucleotide sequence specificallyhybridizes to a target sequence (the nucleotide sequence of any of thenucleic acids described herein) in an amount that is detectably strongerthan non-specific hybridization. High stringency conditions includeconditions which would distinguish a polynucleotide with an exactcomplementary sequence, or one containing only a few scatteredmismatches from a random sequence that happened to have a few smallregions (e.g., 3-10 bases) that matched the nucleotide sequence. Suchsmall regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive CARs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

In an embodiment, the nucleic acids of the invention can be incorporatedinto a recombinant expression vector. In this regard, an embodiment ofthe invention provides recombinant expression vectors comprising any ofthe nucleic acids of the invention. For purposes herein, the term“recombinant expression vector” means a genetically-modifiedoligonucleotide or polynucleotide construct that permits the expressionof an mRNA, protein, polypeptide, or peptide by a host cell, when theconstruct comprises a nucleotide sequence encoding the mRNA, protein,polypeptide, or peptide, and the vector is contacted with the cell underconditions sufficient to have the mRNA, protein, polypeptide, or peptideexpressed within the cell. The vectors of the invention are notnaturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. The inventive recombinant expression vectors cancomprise any type of nucleotides, including, but not limited to DNA andRNA, which can be single-stranded or double-stranded, synthesized orobtained in part from natural sources, and which can contain natural,non-natural or altered nucleotides. The recombinant expression vectorscan comprise naturally-occurring or non-naturally-occurringinternucleotide linkages, or both types of linkages. Preferably, thenon-naturally occurring or altered nucleotides or internucleotidelinkages do not hinder the transcription or replication of the vector.In an embodiment of the invention, the recombinant expression vectorcomprising the nucleotide sequence encoding the fCD27-CD3ζ (CAR does notencode one or both of truncated CD19 and DsRed.

In an embodiment, the recombinant expression vector of the invention canbe any suitable recombinant expression vector, and can be used totransform or transfect any suitable host cell. Suitable vectors includethose designed for propagation and expansion or for expression or both,such as plasmids and viruses. The vector can be selected from the groupconsisting of the pUC series (Fermentas Life Sciences, Glen Burnie,Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pETseries (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).Bacteriophage vectors, such as λGT10, λGT11, λZap11 (Stratagene),λEMBL4, and λNM1149, also can be used. Examples of plant expressionvectors include pB101, pB1101.2, pB1101.3, pB1121 and pBIN19 (Clontech).Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo(Clontech). The recombinant expression vector may be a viral vector,e.g., a retroviral vector or a lentiviral vector. In some embodiments,the vector can be a transposon.

In an embodiment, the recombinant expression vectors of the inventioncan be prepared using standard recombinant DNA techniques described in,for example, Green and Sambrook, supra. Constructs of expressionvectors, which are circular or linear, can be prepared to contain areplication system functional in a prokaryotic or eukaryotic host cell.Replication systems can be derived, e.g., from ColEl, 2μ plasmid, λ,SV40, bovine papilloma virus, and the like.

The recombinant expression vector may comprise regulatory sequences,such as transcription and translation initiation and termination codons,which are specific to the type of host cell (e.g., bacterium, fungus,plant, or animal) into which the vector is to be introduced, asappropriate, and taking into consideration whether the vector is DNA- orRNA-based. The recombinant expression vector may comprise restrictionsites to facilitate cloning.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostto provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the CAR, orto the nucleotide sequence which is complementary to or which hybridizesto the nucleotide sequence encoding the CAR. The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, or a promoter found in the long-terminalrepeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Further, the recombinant expression vectors can be made to include asuicide gene. As used herein, the term “suicide gene” refers to a genethat causes the cell expressing the suicide gene to die. The suicidegene can be a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

An embodiment of the invention further provides a host cell comprisingany of the recombinant expression vectors described herein. As usedherein, the term “host cell” refers to any type of cell that can containthe inventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5α E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell may be a prokaryoticcell, e.g., a DH5α cell. For purposes of producing a recombinant CAR,the host cell may be a mammalian cell. The host cell may be a humancell. While the host cell can be of any cell type, can originate fromany type of tissue, and can be of any developmental stage, the host cellmay be a peripheral blood lymphocyte (PBL) or a peripheral bloodmononuclear cell (PBMC). The host cell may be a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. The T cell may be a human T cell. The T cell may be a Tcell isolated from a human. The T cell can be any type of T cell and canbe of any developmental stage, including but not limited to, CD4⁺/CD8⁺double positive T cells, CD4⁺ helper T cells, e.g., Th₁ and Th₂ cells,CD8⁺ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memoryT cells, naïve T cells, and the like. The T cell may be a CD8⁺ T cell ora CD4⁺ T cell.

Also provided by an embodiment of the invention is a population of cellscomprising at least one host cell described herein. The population ofcells can be a heterogeneous population comprising the host cellcomprising any of the recombinant expression vectors described, inaddition to at least one other cell, e.g., a host cell (e.g., a T cell),which does not comprise any of the recombinant expression vectors, or acell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, anerythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, amuscle cell, a brain cell, etc. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly host cells (e.g., consisting essentially of) comprisingthe recombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

In an embodiment of the invention, the numbers of cells in thepopulation may be rapidly expanded. Expansion of the numbers of cellsexpressing the CAR can be accomplished by any of a number of methods asare known in the art as described in, for example, U.S. Pat. Nos.8,034,334; 8,383,099; U.S. Patent Application Publication No.2012/0244133; Dudley et al., J. Immunother., 26:332-42 (2003); andRiddell et al., J. Immunol. Methods, 128:189-201 (1990). In anembodiment, expansion of the numbers of cells is carried out byculturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g.,irradiated allogeneic PBMC).

CARs, nucleic acids, recombinant expression vectors, and host cells(including populations thereof), all of which are collectively referredto as “inventive CAR materials” hereinafter, can be isolated and/orpurified. The term “isolated” as used herein means having been removedfrom its natural environment. The term “purified” or “isolated” does notrequire absolute purity or isolation; rather, it is intended as arelative term. Thus, for example, a purified (or isolated) host cellpreparation is one in which the host cell is more pure than cells intheir natural environment within the body. Such host cells may beproduced, for example, by standard purification techniques. In someembodiments, a preparation of a host cell is purified such that the hostcell represents at least about 50%, for example at least about 70%, ofthe total cell content of the preparation. For example, the purity canbe at least about 50%, can be greater than about 60%, about 70% or about80%, or can be about 100%.

The inventive CAR materials can be formulated into a composition, suchas a pharmaceutical composition. In this regard, an embodiment of theinvention provides a pharmaceutical composition comprising any of theCARs, nucleic acids, expression vectors, and host cells (includingpopulations thereof), and a pharmaceutically acceptable carrier. Theinventive pharmaceutical compositions containing any of the inventiveCAR materials can comprise more than one inventive CAR material, e.g., aCAR and a nucleic acid, or two or more different CARs. Alternatively,the pharmaceutical composition can comprise an inventive CAR material incombination with other pharmaceutically active agents or drugs, such aschemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc. In a preferred embodiment, the pharmaceuticalcomposition comprises the inventive host cell or populations thereof.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used for the particular inventive CAR material underconsideration. Such pharmaceutically acceptable carriers are well-knownto those skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichhas no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive CAR material, as well as by the particular method used toadminister the inventive CAR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Suitable formulations may include any of those for oral,parenteral, subcutaneous, intravenous, intramuscular, intraarterial,intrathecal, or interperitoneal administration. More than one route canbe used to administer the inventive CAR materials, and in certaininstances, a particular route can provide a more immediate and moreeffective response than another route.

Preferably, the inventive CAR material is administered by injection,e.g., intravenously. When the inventive CAR material is a host cellexpressing the inventive CAR, the pharmaceutically acceptable carrierfor the cells for injection may include any isotonic carrier such as,for example, normal saline (about 0.90% w/v of NaCl in water, about 300mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOLR electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter,Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In anembodiment, the pharmaceutically acceptable carrier is supplemented withhuman serum albumen.

The dose of the inventive CAR material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive CAR material.Typically, the attending physician will decide the dosage of theinventive CAR material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive CAR material to beadministered, route of administration, and the severity of the cancerbeing treated. In an embodiment in which the inventive CAR material is apopulation of cells, the number of cells administered per infusion mayvary, e.g., from about 1×10⁶ to about 1×10¹² cells or more. In certainembodiments, fewer than 1×10⁶ cells may be administered.

For purposes of the invention, the amount or dose of the inventive CARmaterial administered should be sufficient to effect a therapeutic orprophylactic response in the subject or animal over a reasonable timeframe. For example, the dose of the inventive CAR material should besufficient to bind to antigen, or detect, treat or prevent cancer in aperiod of from about 2 hours or longer, e.g., about 12 to about 24 ormore hours, from the time of administration. In certain embodiments, thetime period could be even longer. The dose will be determined by theefficacy of the particular inventive CAR material and the condition ofthe animal (e.g., human), as well as the body weight of the animal(e.g., human) to be treated.

For purposes of the invention, an assay, which comprises, for example,comparing the extent to which target cells are lysed and/or IFN-γ issecreted by T cells expressing the inventive CAR upon administration ofa given dose of such T cells to a mammal, among a set of mammals ofwhich is each given a different dose of the T cells, could be used todetermine a starting dose to be administered to a mammal. The extent towhich target cells are lysed and/or IFN-γ is secreted uponadministration of a certain dose can be assayed by methods known in theart.

One of ordinary skill in the art will readily appreciate that theinventive CAR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive CAR materials is increased through the modification. Forinstance, the inventive CAR materials can be conjugated either directlyor indirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., inventive CAR materials, to targetingmoieties is known in the art.

When the inventive CAR materials are administered with one or moreadditional therapeutic agents, one or more additional therapeutic agentscan be coadministered to the mammal. By “coadministering” is meantadministering one or more additional therapeutic agents and theinventive CAR materials sufficiently close in time such that theinventive CAR materials can enhance the effect of one or more additionaltherapeutic agents, or vice versa. In this regard, the inventive CARmaterials can be administered first and the one or more additionaltherapeutic agents can be administered second, or vice versa.Alternatively, the inventive CAR materials and the one or moreadditional therapeutic agents can be administered simultaneously. Anexemplary therapeutic agent that can be co-administered with the CARmaterials is IL-2. It is believed that IL-2 enhances the therapeuticeffect of the inventive CAR materials.

It is contemplated that the inventive pharmaceutical compositions, CARs,nucleic acids, recombinant expression vectors, host cells, orpopulations of cells can be used in methods of treating or preventingcancer in a mammal. Without being bound to a particular theory ormechanism, the inventive CARs have biological activity, e.g., ability torecognize antigen, e.g., CD70, such that the CAR when expressed by acell is able to mediate an immune response against the cell expressingthe antigen, e.g., CD70, for which the CAR is specific. In this regard,an embodiment of the invention provides a method of treating orpreventing cancer in a mammal, comprising administering to the mammalthe CARs, the nucleic acids, the recombinant expression vectors, thehost cells, the population of cells, and/or the pharmaceuticalcompositions of the invention in an amount effective to treat or preventcancer in the mammal.

An embodiment of the invention further comprises lymphodepleting themammal prior to administering the inventive CAR materials. Examples oflymphodepletion include, but may not be limited to, nonmyeloablativelymphodepleting chemotherapy, myeloablative lymphodepletingchemotherapy, total body irradiation, etc.

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

The mammal referred to herein can be any mammal. As used herein, theterm “mammal” refers to any mammal, including, but not limited to,mammals of the order Rodentia, such as mice and hamsters, and mammals ofthe order Logomorpha, such as rabbits. The mammals may be from the orderCarnivora, including Felines (cats) and Canines (dogs). The mammals maybe from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). Themammals may be of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). Preferably, the mammal is ahuman.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma),bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancerof the anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, headand neck cancer (e.g., head and neck squamous cell carcinoma),glioblastoma, Hodgkin's lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g.,non-small cell lung carcinoma), lymphoma, diffuse large-B-cell lymphoma,follicular lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin's lymphoma (NHL),B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocyticleukemia (ALL), and Burkitt's lymphoma, ovarian cancer, pancreaticcancer, peritoneum, omentum, and mesentery cancer, pharynx cancer,prostate cancer, RCC, ccRCC, rectal cancer, renal cancer, skin cancer,small intestine cancer, soft tissue cancer, solid tumors, stomachcancer, testicular cancer, thyroid cancer, and ureter cancer.Preferably, the cancer is characterized by the expression of CD70. In apreferred embodiment, the cancer is any of RCC (for example, ccRCC),glioblastoma, NHL, CLL, diffuse large-B-cell lymphoma, and follicularlymphoma.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

Another embodiment of the invention provides a use of the inventiveCARs, nucleic acids, recombinant expression vectors, host cells,populations of cells, or pharmaceutical compositions, for the treatmentor prevention of cancer in a mammal.

Another embodiment of the invention provides a method of detecting thepresence of cancer in a mammal, comprising: (a) contacting a samplecomprising one or more cells from the mammal with the CARs, the nucleicacids, the recombinant expression vectors, the host cells, or thepopulation of cells, of the invention, thereby forming a complex, (b)and detecting the complex, wherein detection of the complex isindicative of the presence of cancer in the mammal.

The sample may be obtained by any suitable method, e.g., biopsy ornecropsy. A biopsy is the removal of tissue and/or cells from anindividual. Such removal may be to collect tissue and/or cells from theindividual in order to perform experimentation on the removed tissueand/or cells. This experimentation may include experiments to determineif the individual has and/or is suffering from a certain condition ordisease-state. The condition or disease may be, e.g., cancer.

With respect to an embodiment of the inventive method of detecting thepresence of cancer in a mammal, the sample comprising cells of themammal can be a sample comprising whole cells, lysates thereof, or afraction of the whole cell lysates, e.g., a nuclear or cytoplasmicfraction, a whole protein fraction, or a nucleic acid fraction. If thesample comprises whole cells, the cells can be any cells of the mammal,e.g., the cells of any organ or tissue, including blood cells orendothelial cells.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive CARs, polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells, orpopulations of cells, described herein, can be labeled with a detectablelabel such as, for instance, a radioisotope, a fluorophore (e.g.,fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g.,alkaline phosphatase, horseradish peroxidase), and element particles(e.g., gold particles).

Methods of testing a CAR for the ability to recognize target cells andfor antigen specificity are known in the art. For instance, Clay et al.,J. Immunol., 163: 507-513 (1999), teaches methods of measuring therelease of cytokines (e.g., interferon-γ, granulocyte/monocyte colonystimulating factor (GM-CSF), tumor necrosis factor a (TNF-α) orinterleukin 2 (IL-2)). In addition, CAR function can be evaluated bymeasurement of cellular cytoxicity, as described in Zhao et al., J.Immunol., 174: 4415-4423 (2005).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the transduction efficiency of a retroviralvector encoding a CAR including full-length mouse CD27 and a mouse CD3ζT cell intracellular signaling domain (mCD27-CD3ζ CAR) and having theamino acid sequence of SEQ ID NO: 25 and the reactivity of themCD27-CD3ζ (CAR against mCD70-expressing tumor cells in vitro.

A retroviral vector encoding a CAR including full-length mouse CD27 anda mouse CD3ζ T cell intracellular signaling domain (mCD27-CD3ζ CAR) andhaving the amino acid sequence of SEQ ID NO: 25 was constructed. MurineT cells were retrovirally transduced with the mCD27-CD3ζ (CAR retroviralvector. Transduction efficiency was determined to be 62.6%.

Mouse T cells generated from splenocytes were untransduced (UT) ortransduced with a vector encoding GFP or the mCD27-CD3ζ CAR (effectorcells) and cultured alone (medium) or co-cultured with B16 melanomacells that do not express mouse CD70 (B16 cells) or B16 cells that weretransduced to express mouse CD70 (B16/mCD70) target cells. Pmel cells,which are mouse T cells that recognize B16 tumor, were used as apositive control effector cell. IFN-γ secretion was measured. Theresults are shown in Table 2. As shown in Table 2, cells transduced withthe mCD27-CD3ζ (CAR showed high reactivity against CD70-expressingtumors in vitro.

TABLE 2 IFN-γ (pg/ml) B16 B16/mCD70 Medium Medium 0 0 0 pmel 795 1762 0Mouse T cells/UT 0 0 0 Mouse T 0 0 0 cells/mGFP Mouse T cells/ 0 6421220 mCD27-CD3ζ CAR

Example 2

This example demonstrates that mouse T cells generated from splenocytestransduced with a nucleotide sequence encoding a CAR includingfull-length mouse CD27 and a mouse CD3ζ T cell intracellular signalingdomain (mCD27-CD3ζ CAR) and having the amino acid sequence of SEQ ID NO:25 reduces tumor burden and increases the survival of CD70-expressingtumor-bearing mice.

Eleven days prior to transferring CAR-expressing cells into mice, tumorswere established in mice by injecting them with B16 cells or B16/mCD70cells. Four days later, splenocytes were removed from the mice andstimulated with concanavalin A (ConA) and IL-7 or anti-mouse CD3 (mCD3)and soluble CD28 (sCD28). Two days later, mouse T cells generated fromthe stimulated splenocytes were transduced with a MSGV1 retroviralvector encoding the mCD27-CD3ζ CAR having the amino acid sequence of SEQID NO: 25. Five days later, the mCD27-CD3ζ CAR-transduced cells (1×10⁷)were administered to the tumor-bearing mice, and the mice wereirradiated (500 rads). Control tumor-bearing mice were administereduntransduced cells, phosphate buffered saline (PBS), or a combination ofpmel cells (pmel), a gp100 vaccine (V), and IL-2 (I) (“pmel+VI”) andirradiated. The size of the tumors was measured over a period of time upto about 35 days after treatment. The results are shown in FIGS. 1A-1B.As shown in FIGS. 1A-1B, the mCD27-CD3ζ CAR-transduced cells reduced thetumor burden in B16/mCD70-tumor bearing mice, but not in B16-tumorbearing mice. Accordingly, mice bearing CD70+ tumors could besuccessfully treated with mCD27-CD3ζ CAR-transduced cells, and thetreatment was CD70-specific.

The experiment was repeated with B16/mCD70-tumor bearing mice, exceptthat splenocytes were stimulated with anti-mCD3 and sCD28, and the micewere also administered IL-2 after irradiation and administration oftransduced cells. Control tumor-bearing mice were administereduntransduced cells, cells transduced with an empty vector, or pmel+VI,followed by irradiation and administration of IL-2. The size of thetumors was measured over a period of time up to about 24 days aftertreatment. The results are shown in FIG. 1F. As shown in FIG. 1F, whenco-administered with IL-2, the mCD27-CD3ζ CAR-transduced cells reducedthe tumor burden in B16/mCD70-tumor bearing mice.

Twenty-one days after cell transfer, the tumors were removed from thetreated mice and grown in vitro for seven days. Mouse CD70 expression inthe tumors was measured by FACS. It was observed that CD70 expressionwas lost in mice treated with mCD27-CD3ζ (CAR-transduced cells but notin mice treated with Pmel+V or untransduced cells. Without being boundto a particular theory or mechanism, it is believed that recurrence oftumor growth was most likely due to the loss of CD70 expression onB16/mCD70 tumors.

The experiment corresponding to that of FIG. 1B was repeated again withB16/mCD70 tumor-bearing mice, with the following exceptions. B16/mCD70tumor-bearing mice were administered mCD27-CD3ζ (CAR-transduced cells ata dose of 1×10⁴, 1×10⁵, 1×10⁶, or 1×10⁷ cells per mouse with or withoutirradiation (500 Rads). Control tumor-bearing mice were administeredPBS, pmel+VI, or mouse T cells that were transduced with an empty vectorwith or without irradiation (500 Rads). The results are shown in FIGS.1C-1D. As shown in FIG. 1C, the lowest effective dose for treatingtumors was 1×10⁵ mCD27-CD3ζ CAR-transduced cells per mouse when micewere irradiated. As shown in FIGS. 1C-1D, at a dose of 1×10⁷ cells permouse, irradiation did not seem to affect treatment efficacy.

Survival of the tumor-bearing mice was also assessed over a period oftime up to about 42 days after treatment. The results are shown in FIG.1E. As shown in FIG. 1E, irradiated tumor-bearing mice treated with themCD27-CD3ζ CAR-transduced cells survived longer, particularly at dosesof 1×10⁶ or 1×10⁷ cells per mouse.

Example 3

This example demonstrates that administration of cells transduced withthe mCD27-CD3ζ CAR to tumor-bearing mice results in some toxicity. Thisexample also demonstrates that the mice can recover from the toxicity.

B16 or B16/mCD70-tumor bearing mice were administered untransduced cellsor cells transduced with the mCD27-CD3ζ (CAR having the amino acidsequence of SEQ ID NO: 25, PBS, or pmel+V, with or without irradiation(500 Rads). The average weight of the mice was measured over a periodbeginning about six days after cell transfer up to about 17 daysfollowing treatment. The results are shown in FIGS. 2A-2D. As shown inFIGS. 2A-2D, transient lower body weights were observed for bothB16/mCD70 and B16-tumor bearing mice that were treated with themCD27-CD3ζ CAR. The lower body weight observed in the mCD27-CD3ζCAR-treated mice was irrelevant to implanted tumors. Without being boundto a particular theory or mechanism, it is believed that the lower bodyweight implies that endogenous cells were targeted by the mCD27-CD3ζCAR. The mice recovered lost body weight when they were administered ahydrogel containing water, hydrocolloids, food acid, and sodiumbenzoate.

B16 or B16/mCD70-tumor bearing mice were administered untransduced cellsor cells transduced with the mCD27-CD3ζ CAR having the amino acidsequence of SEQ ID NO: 25, or cells transduced with a vector encodingGFP, with or without irradiation (500 Rads). The absolute white bloodcell (WBC) count in the mice was measured over a period beginning aboutsix days after cell transfer up to about 14 days following treatment.The results are shown in FIGS. 2E-2H. As shown in FIGS. 2E-2F, atransient lower WBC count was observed in the mice that were treatedwith the mCD27-CD3ζ (CAR. As shown in FIGS. 2G-2H, a transient lowersplenocyte count was also observed in the mice that were treated withthe mCD27-CD3ζ CAR.

B16/mCD70-tumor bearing mice were administered cells transduced with themCD27-CD3ζ (CAR having the amino acid sequence of SEQ ID NO: 25 or cellstransduced with a vector encoding GFP, with or without irradiation (500Rads). Serum IFN-γ levels were measured for a period beginning aboutthree days after cell transfer up to about seven days after treatment.The results are shown in FIG. 2I. As shown in FIG. 2I, transient IFN-γsecretion was observed in the irradiated mice treated with themCD27-CD3ζ (CAR.

Example 4

This example demonstrates that administration of the mCD27-CD3ζ (CARdoes not have a measurable effect on the long-term immune function ofnon-tumor bearing mice.

Non-tumor bearing mice were administered cells that were transduced withthe mCD27-CD3ζ CAR having the amino acid sequence of SEQ ID NO: 25 orcells transduced with a vector encoding GFP (GFP), with or withoutirradiation (500 Rads). The mice were immunized with ovalbumin (OVA) orhuman (h) gp100 32 or 50 days after transfer of transduced cells. Tcells were removed from the spleen and lymph node (LN) of the mice sevendays after immunization. The cells were stimulated in vitro with OT-1,OT-II, or hgp100 peptide. The results are shown in Table 3 (Day32-spleen), Table 4 (Day 32-LN), and Table 5 (Day 50-spleen). In Table5, immunized C57BL/6 (immune competent) mice (B6/Im) were used as apositive control. Naïve C57BL/6 mice (B6/naive) were used as a negativecontrol. As shown in Tables 3-5, administration of the mCD27-CD3ζ (CARdoes not have a measurable effect on the long-term immune function ofnon-tumor bearing mice.

The histology of various organs, including brain, lung, liver, kidney,intestine, heart, spleen, and bone were examined from 3 days to 7 daysafter cell transfer. The chemistry of the blood, in particular, theblood levels of sodium, potassium, chloride, calcium, magnesium,phosphorus, glucose, blood urea nitrogen (BUN), creatinine, uric acid,albumin, protein, cholesterol, triglycerides, alkaline phosphatase (ALKP), alanineaminotransferase (ALT/GPT), aspartate aminotransferase(AST/GOT), amylase, creatine kinase (CK), and lactate dehydrogenase (LD)were examined from 3 days to 7 days after cell transfer. No changes inhistology or blood chemistry were observed.

TABLE 3 Immunized with: OVA hgp100 Stimulated With: OT-1 OT-II hgp100mCD27- 2263 54 44 48 1293  <32 CD3ζ CAR (500 Rads) GFP (500 Rads) 113084 67 60 177 40 mCD27-  347 <32 <32 <32 298 <32 CD3ζ CAR GFP  933 96 9693 544 80

TABLE 4 Immunized with: OVA hgp100 Stimulated With: OT-1 OT-II hgp100mCD27- <32 <32 12980  <32 <35 <32 CD3ζ CAR (500 Rads) GFP (500 Rads)  62<32 230 <32 <35 45 mCD27- 235 66 557 32 301 139 CD3ζ CAR GFP 121 <32 34035 <35 <32

TABLE 5 Immunized with: OVA hgp100 Stimulated With: OT-1 OT-II hgp100mCD27- 1708 24 575 <32 <32 <32 CD3ζ CAR (500 Rads) GFP (500 Rads)  498114 4429  122 <32 <32 mCD27- 1219 77 995 <32 <32 <32 CD3ζ CAR GFP  371<32 370 <32  67 <32 B6/lm 1138 79 245 39 119 33 B6/naive  <32 <32 134 70<32 <32

Example 5

This example demonstrates that T cells transduced with a nucleotidesequence encoding a CAR including full-length human CD27 and a humanCD3ζ (T cell intracellular signaling domain (fCD27-CD3ζ (CAR) expressthe CAR following expansion of the numbers of transduced cells.

PBL were non-specifically stimulated with OKT3 and T cells generatedfrom the PBL were (a) untransduced (UT), (b) transduced with anucleotide sequence encoding full-length human CD27 (fCD27), or (c)transduced with a nucleotide sequence encoding the fCD27-CD3ζ CAR havingthe amino acid sequence of SEQ ID NO: 7. The cells were grown, analyzedfor CAR expression by fluorescence-activated cell sorting (FACS), andtested for tumor reactivity based on IFN-γ production. The numbers ofCD70-reactive cells were rapidly expanded (REP) generally as describedin Riddell et al., J. Immunol. Methods, 128: 189-201 (1990). Expandednumbers of cells were grown and analyzed for expression of CD27, CD70,CD45RO, and CD62L by FACS. Table 6 shows the percentage of cells withthe indicated phenotypes as measured by FACS. Table 7 shows the foldexpansion and viability (%) of the cells following stimulation (butbefore REP) and after REP. As shown in Tables 6 and 7, expanded numbersof transduced cells express the CAR and are viable and have an effectormemory phenotype.

TABLE 6 fCD27-CD3ζ UT fCD27 CAR After CD27+/CD70+ 10.84% 2.04% 3.27%stimulation CD27−/CD70+ 25.85% 0.53% 0.36% and before CD27+/CD70− 44.82%85.18% 94.55% REP CD27−/CD70− 18.49% 12.24% 1.82% CD45RO+/CD62L+ 48.97%29.02% 39.77% CD45RO−/CD62L+  8.14% 7.38% 6.50% CD45RO+/CD62L− 31.70%48.63% 47.27% CD45RO−/CD62L− 11.19% 14.97% 6.47% After REP CD27+/CD70+ 5.20% 4.93% 0.45% CD27−/CD70+ 70.02% 12.53% 0.12% CD27+/CD70− 11.01%71.76% 91.84% CD27−/CD70− 13.77% 10.78% 7.59% CD45RO+/CD62L+ 17.42%14.52% 12.39% CD45RO−/CD62L+  2.40% 1.59% 5.53% CD45RO+/CD62L−  72.9%73.98% 44.37% CD45RO−/CD62L−  7.40% 9.90% 37.70%

TABLE 7 Fold expansion Viability (%) After stimulation UT 6 88 andbefore REP fCD27 3 84 fCD27-CD3ζ CAR 3 70 After REP UT 720 86 fCD27 56075 fCD27-CD3ζ CAR 790 79

Example 6

This example demonstrates that T cells transduced with a nucleotidesequence encoding a CAR including full-length human CD27 and a humanCD3ζ (T cell intracellular signaling domain (fCD27-CD3ζ (CAR)proliferate upon co-culture with CD70-expressing cells and specificallyrecognize CD70-expressing tumor cell lines in vitro.

T cells were generated from human PBL. Untransduced (UT) T cells or Tcells transduced with a nucleotide sequence encoding fCD27 or thefCD27-CD3ζ CAR (effector cells) were cultured alone or co-cultured withCD70-expressing tumor cell line 624 mel or 624 mel cells transduced withCD70 (624/CD70) (target cells). Proliferation of the effector cells wasmeasured using carboxyfluorescein succinimidyl ester (CFSE) on day 4 ofthe co-culture. The T cells transduced with the fCD27-CD3ζ CARproliferated only when co-cultured with the CD70-expressing tumor cellline 624/CD70. The UT T cells and the T cells transduced with fCD27 didnot proliferate in any culture.

UT T cells or T cells transduced with a nucleotide sequence encodingfCD27 or the fCD27-CD3ζ (CAR (SEQ ID NO: 7) (effector cells) werecultured alone (medium) or co-cultured with one of the human RCC celllines or control cell lines 624, 624/CD70, SNU245, SNU1079, or SNU1196(target cells) shown in Table 8 below. All SNU cell lines were CD70negative. IFN-γ secretion was measured. The results are shown in Table8. As shown in Table 8, T cells transduced with a nucleotide sequencethe fCD27-CD3ζ (CAR (SEQ ID NO: 7) were reactive against CD70-expressinghuman RCC cell lines.

TABLE 8 IFN-γ (pg/ml) CD70 fCD27-CD3ζ expression UT fCD27 CAR 624Negative 39 170 87 (Neg) 624/CD70 Positive (Pos) 29 173 6485 RCC 2219RPos 61 147 12068 RCC 2245R Pos 29 103 9108 RCC 2095R Pos 40 210 5819 RCC1581 Pos 41 163 11797 RCC 2246R Pos 27 94 8221 RCC 2657R Pos 17 48 3510RCC 2361R Pos 14 48 2256 RCC 2261R Pos 60 129 7267 RCC 2654R Pos 38 1507894 SNU245 Neg 86 150 36 SNU1079 Neg 70 110 33 SNU1196 Neg 35 85 25Medium — 185 389 53

Example 7

This example demonstrates the transduction efficiency of anti-CD70 humanCAR constructs.

Human T cells were transduced with an empty retroviral vector (Mock) ora retroviral vector encoding one of the constructs set forth in Tables9A-9C. CARs including a truncated (Δ) CD27 lack all of the CD27intracellular T cell signaling domain, that is, the truncated CD27 lackscontiguous amino acid residues 212-260 of SEQ ID NO: 2. Transduced cellswere analyzed for CD3, CD27, CD62L, and CD45RO expression by FACS.Tables 9A-9C show the percentage of cells with the indicated phenotypesas measured by FACS. As shown in Table 9B, cells transduced with CARshave an effector memory phenotype.

TABLE 9A Phenotype (%) CD3+/ CD3+/ CD3−/ CD27+ CD27− CD3−/CD27+ CD27−full length (f) CD27-CD3ζ 75.90 5.85 16.00 2.23 (SEQ ID NO: 7) truncated(Δ) CD27-CD28- 44.30 51.60 0.70 3.41 CD3ζ (SEQ ID NO: 8)ΔCD27-4-1BB-CD3ζ 60.20 27.90 8.16 3.71 (SEQ ID NO: 9) ΔCD27-CD28-4-1BB-16.0 80.30 0.30 3.39 CD3ζ (SEQ ID NO: 10) fCD27-CD28-CD3ζ 3.11 93.900.037 2.98 (SEQ ID NO: 11) fCD27-4-1BB-CD3ζ 60.70 29.20 5.98 4.10 (SEQID NO: 12) fCD27-CD28-4-1BB- 60.80 23.40 9.66 6.08 CD3ζ (SEQ ID NO: 13)Mock (control) (empty 0.26 97.50 6.75 × 10⁻³ 2.28 vector)

TABLE 9B Phenotype (%) CD45RO+/ CD45RO+/ CD45RO−/ CD45RO−/ CD62L+ CD62L−CD62L+ CD62L− full length (f) CD27-CD3ζ 68.30 23.50 6.13 2.03 (SEQ IDNO: 7) truncated (Δ) CD27-CD28- 84.70 9.52 4.49 1.25 CD3ζ (SEQ ID NO: 8)ΔCD27-4-1BB-CD3ζ (SEQ 74.00 20.20 4.20 1.60 ID NO: 9) ΔCD27-CD28-4-1BB-86.40 7.83 4.54 1.19 CD3ζ (SEQ ID NO: 10) fCD27-CD28-CD3ζ (SEQ 87.709.72 1.76 0.86 ID NO: 11) fCD27-4-1BB-CD3ζ (SEQ 69.10 22.70 5.80 2.35 IDNO: 12) fCD27-CD28-4-1BB- 73.20 13.90 10.20 2.72 CD3ζ (SEQ ID NO: 13)Mock (control) (empty 85.50 11.60 2.11 0.73 vector)

TABLE 9C Phenotype (%) CD27+/ CD27+/ CD27−/ CD70+ CD70− CD27−/CD70+CD70− full length (f) CD27-CD3ζ 1.63 96.40 0.10 1.89 (SEQ ID NO: 7)truncated (Δ) CD27-CD28- 0.69 90.80 0.46 8.06 CD3ζ (SEQ ID NO: 8)ΔCD27-4-1BB-CD3ζ (SEQ 0.77 95.40 0.057 3.77 ID NO: 9) ΔCD27-CD28-4-1BB-0.42 83.80 0.66 15.10 CD3ζ (SEQ ID NO: 10) fCD27-CD28-CD3ζ (SEQ 9.3068.10 11.60 11.0 ID NO: 11) fCD27-4-1BB-CD3ζ (SEQ 1.09 92.20 0.16 6.55ID NO: 12) fCD27-CD28-4-1BB- 2.04 92.70 0.11 5.18 CD3ζ (SEQ ID NO: 13)Mock (control) (empty 1.67 28.50 51.30 18.50 vector)

Example 8

This example demonstrates that human T cells transduced with f CD27-CD3ζ(SEQ ID NO: 7), ΔCD27-4-1BB-CD3ζ (SEQ ID NO: 9), ΔCD27-CD28-4-1BB-CD3ζ(SEQ ID NO: 10), fCD27-4-1BB-CD3ζ (SEQ ID NO: 12), orfCD27-CD28-4-1BB-CD3ζ (SEQ ID NO: 13) recognize CD70-expressing RCCtumor cells in vitro.

Human T cells were transduced with an empty retroviral vector (MSGV1) ora retroviral vector encoding one of the constructs set forth in Table9A. Transduced cells were cultured alone (medium) or co-cultured withcontrol target cells 624 mel, 624/CD70, 938 mel, or 938 mel cellstransduced to express CD70 (938/CD70) or RCC target cells RCC 2245R, RCC2246R, RCC 2361R, or RCC 1764. IFN-γ secretion was measured. The resultsare shown in FIG. 3. As shown in FIG. 3, human T cells transduced withfCD27-CD3ζ (SEQ ID NO: 7), ΔCD27-4-1BB-CD3ζ (SEQ ID NO: 9),ΔCD27-CD28-4-1BB-CD3ζ (SEQ ID NO: 10), fCD27-4-1BB-CD3ζ (SEQ ID NO: 12),or fCD27-CD28-4-1BB-CD3ζ (SEQ ID NO: 13) recognize CD70-expressing RCCtumor cells in vitro.

Example 9

This example demonstrates the selection of a ΔCD27-4-1BB-CD3ζ ((SEQ IDNO: 9) retroviral-vector producing packaging clone.

Retroviral packaging cell line PG13 clones A2, A10, B3, C1, E3, G2, wereuntransduced or transduced with a retroviral vector encodingΔCD27-4-1BB-CD3ζ ((SEQ ID NO: 9). Table 10 shows the percentage of cellswith the indicated phenotypes as measured by FACS.

TABLE 10 Phenotype (%) CD3−/ CD3+/ CD3+/CD27+ CD27+ CD27− CD3−/CD27− A232.6 0.30 65.9 1.16 A10 31.0 0.34 67.7 0.94 B3 27.6 0.25 71.2 0.91 C130.0 0.33 68.7 0.94 E3 40.9 0.40 57.8 0.95 G2 18.6 0.17 80.3 0.95Untransduced (UT) 0.12 0.020 98.6 1.28

The transduced clones were cultured alone (medium) or co-cultured withtarget control cells 938 mel, 938/CD70, SNU1079, SNU1196, or target RCCcell lines RCC 2245R, RCC 2246R, RCC 2361R, or RCC 1764. IFN-γ secretionwas measured. The results are shown in FIG. 4. As shown in FIG. 4,retroviral packaging clone E3 demonstrated reactivity againstCD70-expressing target tumor cell lines.

Retroviral packaging cell clones were transduced with a CAR as set forthin Table 11. Table 11 shows the percentage of cells with the indicatedphenotypes as measured by FACS.

TABLE 11 Phenotype (%) CD3+/ CD3+/ CD27+ CD3−/CD27+ CD27− CD3−/CD27−PG13/B11/fCD27- 73.5 1.37 24.5 0.62 CD3ζ (SEQ ID NO: 7) PG13/A2/ΔCD27-4-34.7 0.57 63.5 1.18 1BB-CD3ζ (SEQ ID NO: 9) PG13/E3/ΔCD27-4- 50.7 1.2347.0 1.07 1BB-CD3ζ (SEQ ID NO: 9) PG13/C5/fCD27- 45.7 1.48 51.4 1.43CD28-4-1BB- CD3ζ (SEQ ID NO: 13) RD114/D2/fCD27- 30.7 0.65 67.8 0.91CD28-4-1BB- CD3ζ (SEQ ID NO: 13) Untransduced (UT) 0.26 3.45 × 10⁻³ 98.91.71

The transduced clones were cultured alone (medium) or co-cultured withtarget control cells 938 mel, 938/CD70, SNU1079, SNU1196, or target RCCcell lines RCC 2245R, RCC 2246R, RCC 2361R, or RCC 1764. IFN-γ secretionwas measured. The results are shown in FIG. 5. As shown in FIG. 5,retroviral packaging clone E3 demonstrated reactivity againstCD70-expressing target tumor cell lines.

Based on its transduction efficiency and tumor activity, retroviralpackaging clone E3/ΔCD27-4-1BB-CD3ζ (SEQ ID NO: 9) was chosen forclinical use.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A chimeric antigen receptor (CAR) havingantigenic specificity for CD70, the CAR comprising: a CD70binding—transmembrane domain comprising the amino acid sequence of SEQID NO: 3, wherein the CD70 binding—transmembrane domain does notcomprise residues 212 to 260 of the amino acid sequence of SEQ ID NO: 2;a CD3ζ intracellular T cell signaling domain comprising the amino acidsequence of SEQ ID NO: 4; and one or both of (i) a 4-1BB intracellular Tcell signaling domain comprising the amino acid sequence of SEQ ID NO: 5and (ii) a CD28 intracellular T cell signaling domain comprising theamino acid sequence of SEQ ID NO:
 6. 2. The CAR according to claim 1,comprising the 4-1BB intracellular T cell signaling domain and the CD28intracellular T cell signaling domain.
 3. The CAR according to claim 1,comprising the 4-1BB intracellular T cell signaling domain.
 4. The CARof claim 1, comprising the amino acid sequence of any one of SEQ ID NOs:8-10.
 5. A nucleic acid comprising a nucleotide sequence encoding theCAR according to claim
 1. 6. The nucleic acid according to claim 5,comprising the nucleotide sequence of any one of SEQ ID NOs: 15-17.
 7. Arecombinant expression vector comprising the nucleic acid of claim
 6. 8.An isolated host cell comprising the recombinant expression vector ofclaim
 7. 9. An isolated population of cells comprising at least one hostcell of claim
 8. 10. A pharmaceutical composition comprising the CAR ofclaim 1 and a pharmaceutically acceptable carrier.
 11. The CAR of claim4 comprising the amino acid sequence SEQ ID NO:
 8. 12. The CAR of claim4 comprising the amino acid sequence SEQ ID NO:
 9. 13. The CAR of claim4 comprising the amino acid sequence SEQ ID NO:
 10. 14. The nucleic acidaccording to claim 6, comprising the nucleotide sequence of SEQ ID NO:15.
 15. The nucleic acid according to claim 6, comprising the nucleotidesequence of SEQ ID NO:
 16. 16. The nucleic acid according to claim 6,comprising the nucleotide sequence of SEQ ID NO: 17.